Oxidative stress, resulting from increased reactive oxygen species (ROS) generation in the vascular wall, occurs following vascular injury. During this repair process, vascular smooth muscle cell (VSMC) migration across the internal elastic lamina is increased and contributes significantly to neointimal formation. While the increased ROS production following injury is well documented, very little is known regarding the mechanisms by which ROS contribute to VSMC migration and neointimal formation. Recent studies by our group have begun to dissect the molecular signaling mechanisms involved in vascular smooth muscle cell migration that are regulated by ROS and have identified 3-phosphoinositide-dependent kinase-1 (PDK1) as a key regulator. However, we do not yet understand how ROS and/or upstream kinases regulate the cytoskeletal events that control cell chemotaxis, nor whether these novel mechanisms contribute to vascular pathology in vivo. Our working hypothesis is that actin cytoskeletal protrusion at the leading edge during VSMC migration and vascular repair following injury is regulated by the ROS-dependent activation of 3-phosphoinositide-dependent kinase-1 (PDK1) and subsequent activation of a currently unknown protein phosphatase. The following specific aims will be accomplished: 1) determine whether the ROS-dependent activation of PDK1 mediates the dephosphorylation of cofilin to regulate VSMC migration via actin depolymerization and the formation of stress fibers and/or lamellipodia, 2) test if the ROS-dependent activation of PDK1 and its role in cofilin dephosphorylation is due to its localization within specific signaling domains in the cell, and 3) define the contribution of ROS by studying VSMC migration in vivo during vessel remodeling following carotid wire injury in mice. To test these aims we will utilize siRNA, adenoviral vectors, and pharmacological strategies to assess migration and identify key signaling mechanisms in vitro utilizing cultured VSMCs, confocal microscopy of both live and fixed VSMCs to examine cytoskeletal reorganization and protein co-localizations occurring during migration, and an in vivo wire injury model to assess mechanisms by which ROS-mediated VSMC migration contribute to neointimal formation. It is anticipated that the findings from these studies will provide important information about the role of ROS in mediating VSMC migration in vitro and in vivo, and may ultimately identify potential therapeutic targets for intervention during vascular injury and/or disease. Vascular pathologies such as high blood pressure, atherosclerosis, and restenosis and their related complications contribute significantly to mortality in Western cultures. One common component of each of these diseases is that there is some degree of injury which occurs to the blood vessels which typically makes results in further complications requiring treatment and management. During vascular injury the smooth muscle cells, which make up a main layer of the blood vessel structure, move in response to a variety of stimuli. This proposal examines how a well documents change that occurs in response to vascular injury, increased reactive oxygen species production, potentiates the movement of smooth muscle cells. The identification of mechanisms underlying this cell movement will allow for the design of better approaches to treatment of the complications in patients as well as identify targets for the design of pharmacological interventions. Vascular pathologies such as high blood pressure, atherosclerosis, and restenosis and their related complications contribute significantly to mortality in Western cultures. One common component of each of these diseases is that there is some degree of injury which occurs to the blood vessels which typically makes results in further complications requiring treatment and management. During vascular injury the smooth muscle cells, which make up a main layer of the blood vessel structure, move in response to a variety of stimuli. This proposal examines how a well documents change that occurs in response to vascular injury, increased reactive oxygen species production, potentiates the movement of smooth muscle cells. The identification of mechanisms underlying this cell movement will allow for the design of better approaches to treatment of the complications in patients as well as identify targets for the design of pharmacological interventions.